Serial intracerebral passage of dengue type 1 or type 2 virus in mice was shown previously to select for mouse neurovirulent mutants which were attenuated for humans. Later, in a similar manner a neurovirulent mutant of DEN4 (strain H241) was selected by serial intracerebral passage in mice. In addition, DEN4 H241 neurovirulent (N) replicated less efficiently than DEN4 H241 parent (P) in simian LLC-MK2 cells. An intratypic DEN4 chimera containing the C-PreM-E structural protein genes from DEN4 H241N also exhibited marked restriction of growth in LLC-MK2 cells. Analysis of viral proteins produced in LLC-MK2 cells by DEN4 H241N or its derived C-PreM-E chimera indicated that very little PreM was produced and that which was detected migrated slightly slower than the PreM of DEN4 H241P or its chimeric derivative. Recent evidence indicates that immature PreM-containing flavivirus virions are less infectious than the mature M-containing virus. Studies were performed to determine whether altered PreM or mutations in C or E might affect the normal processing of PreM that yields M which is normally the predominant product in the mature virion. Protein analysis indicated that DEN4 E, PreM, M and C were detected in a virion preparation of DEN4 H241P or its derived chimera. On the other hand, a virion preparation of DEN4 H241N or its derived chimera contained E, PreM and C, but not M. This suggested that cleavage of PreM to M was defective for DEN4 H241N and the genetic loci for the defect mapped within the C-PreM-E genes. There are six amino acid differences in the structural protein gene region between DEN4 H241P and DEN4 H241N: one in C, two in PreM and three in E. To identify mutations responsible for defective PreM cleavage, eight mutants were constructed from the intratypic DEN4 P chimeric virus that contained one or more amino acid substitutions that are present in the mutant C, PreM or E. Only mutant DEN4(H241P, S456), into which all three amino acid substitutions present in the E of DEN4 N were introduced, exhibited the PreM cleavage defect. Interestingly, chimeric mutants which contained both mutations in PreM processed PreM normally. This suggests that PreM interacts with E during virus maturation and changes in the latter can have a profound effect on processing of the former protein.